Method for producing xylanase through aspergillus niger fermentation based on response surface methodology optimization

Abstract

The invention provides a method for producing xylanase through aspergillus niger fermentation based on response surface methodology optimization. According to the invention, Aspergillus niger G63 with the preservation number of CGMCC NO.41556 is used as a fermentation strain, so that the high activity of the product and the high efficiency of the production process are ensured. In the aspect of raw material selection, low-cost solid wastes, such as bran and soybean meal, are ingeniously utilized as carbon sources for strain growth, so that cyclic utilization of resources is realized, and the production cost is effectively reduced. In addition, the production process is simple in design, easy to operate and short in fermentation period, and the stability of product quality and the high activity of enzyme are guaranteed. It is worth mentioning that the prepared xylanase shows excellent acid resistance and high temperature resistance, so that the xylanase has wide application prospects and popularization value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation engineering, and particularly relates to a method for producing xylanase by fermenting Aspergillus niger based on response surface methodology optimization. Background Art

[0002] Xylanase, an enzyme of microbial origin, has attracted much attention for its potential for biotechnological applications in multiple industrial fields. The production of this enzyme usually uses lignocellulosic raw materials such as sugarcane bagasse, rice straw, and rice bran, which not only provide the necessary carbon source for production, but also solve the problem of solid waste disposal. Xylanase is mainly prepared by two technologies: solid-state fermentation and submerged fermentation, which are finely regulated by a variety of physical and nutritional conditions.

[0003] In order to achieve the wide application of xylanase in industry, such as biofuel manufacturing, pulp and paper making, food and feed processing, waste paper recycling, etc., researchers have adopted a variety of technologies to optimize its commercial production process to meet the specific needs of economy and industry. Lignocellulose, such as wood, is mainly composed of cellulose and hemicellulose, of which hemicellulose is one of the most abundant renewable resources on earth, and xylan accounts for about 37.2%. Xylan is a polysaccharide connected by β-1,4 glycosidic bonds, and its side chains contain methyl ether and arabinose, etc. These complex structures make xylan difficult to decompose, resulting in waste of resources.

[0004] Xylanases include endo- and exo-xylanases, as well as debranching enzymes that can degrade side chains. The main function of these enzymes is to decompose xylan into oligosaccharides, which have great application potential in many industrial fields, including papermaking, feed, food, brewing, energy conversion, etc. due to their economic value. Summary of the invention

[0005] The present invention aims to overcome the limitations of the prior art and provide a method for efficiently producing xylanase, specifically, a method for producing xylanase by fermenting Aspergillus niger based on response surface methodology optimization.

[0006] The present invention is achieved through the following technical solutions: A method for producing xylanase by fermentation of Aspergillus niger based on response surface methodology optimization, using Aspergillus niger strain Aspergillus sp. G63, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) on October 28, 2024, with a deposit number of CGMCC NO.41556, as the fermentation strain, optimizing the fermentation medium and fermentation conditions based on response surface methodology, and producing xylanase, the specific method comprising the following steps: S1 Single factor experiment to determine the solid fermentation medium and fermentation culture conditions; S2 orthogonal experiment to determine the most relevant influencing factors of solid fermentation medium and fermentation culture conditions; S3, based on orthogonal experiments, selected the most relevant influencing factors and used response surface methodology to optimize solid-state fermentation medium and fermentation culture conditions; S4 prepared xylanase under the solid-state fermentation medium and fermentation conditions optimized by response surface methodology.

[0007] Specifically, in step S1, a single factor experiment is used to investigate the composition of the solid culture medium, wherein the solid culture medium contains a carbon source bran, a nitrogen source soybean meal, an additional nitrogen source, metal ions and water; the experimental factors of the single factor experiment include the ratio of bran to soybean meal (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2), the type of additional nitrogen source (urea, (NH4)2SO4, NH4Cl, KNO3 and NaNO3) and the amount of addition (0.9%, 1.1%, 1.3%, 1.5%, 1.7%), the type of metal ion (K + , Mn 2+ Mg 2+ , Fe 3+ 、Na + ) and addition amount (0.7%, 0.9%, 1.1%, 1.3%, 1.5%), water content (50%, 55%, 60%, 65%, 70% of the total mass of the culture medium).

[0008] In the culture medium obtained by single factor experiment screening, the mass ratio of soybean meal and bran is 3:7; the additional nitrogen source is NH4Cl, and its addition amount is 1.1-1.5%; the metal ion is Na+, and its addition amount is 0.9-1.3%; the water content is 50-60%.

[0009] Furthermore, in the optimal fermentation medium screened by single factor experiment, the mass ratio of soybean meal and bran was 3:7; the additional nitrogen source was NH4Cl, and its addition amount was 1.5%; the metal ion was Na+, and its addition amount was 1.3%; and the water content was 60%.

[0010] Based on the optimal fermentation medium screened by single factor test, single factor test of fermentation culture conditions was further carried out. The test factors included: inoculation volume (0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL); charge volume (10g, 20g, 30g, 40g, 50g); temperature (22℃, 25℃, 28℃, 31℃, 34℃); pH value (3.5, 4.5, 5.5, 6.5, 7.5).

[0011] The fermentation conditions obtained by single factor test screening were: charge volume of 30 g, inoculation volume of 1.5 mL, pH of 5.5, and temperature of 28 °C.

[0012] In step S2, a three-factor three-level orthogonal experimental design was adopted, and the experimental factors of the solid-state fermentation medium were selected: the amount of additional nitrogen source added (1.1%, 1.3%, 1.5%, water content (50%, 55%, 60%) and the amount of metal ion added (0.9%, 1.1%, 1.3%), and the influencing factors most relevant to the culture medium and enzyme activity were analyzed; the experimental factors of the fermentation culture conditions were selected: temperature (25°C, 28°C, 31°C), inoculation size (1.0mL, 1.5mL, 2.0mL), pH (4.5, 5.5, 6.5), and the influencing factors most relevant to the culture conditions and enzyme activity were analyzed.

[0013] Step S3 comprehensively analyzes the results of orthogonal experiments, selects three factors: water content (A), temperature (B) and inoculation amount (C), designs a response surface optimization experiment, and analyzes the experimental results using the Design-Expert software. The fitting equation obtained from the response surface experiment is: Y=26638.3+238.12A+1049.62B+477.75C-350AB-885.25AC+272.25BC-2692.92A²-4437.42B²-2756.67C². The F value in the model equation is 365.78, and the P value is <0.0001, which meets the significant condition of the model. The regression equation determination coefficient R 2 =0.9985, and Adj=0.9958, indicating that the model fits the experiment well, the experimental error is small, and it can better reflect the accurate relationship between the three factors and xylanase in the fermentation process. The influence of each factor on the xylanase activity is B>C>A. AB, AC, and BC all have obvious interactions, especially AC. Therefore, the optimal fermentation medium for enzyme production of the mutant strain Aspergillus niger is: 21g bran, 9g soybean meal, 1.5% NH4Cl addition, and Na + The addition amount was 1.1%, the water content was 60%, the inoculation amount was 1.5 mL, the loading amount was 30 g, the pH was 5.5, the fermentation temperature was 28 ° C, and the culture time was 5 days.

[0014] Step S4, producing xylanase under the solid-state fermentation medium and fermentation conditions optimized by response surface methodology. Experiments have shown that the enzyme activity of the xylanase produced by the solid-state fermentation of the present invention is increased by more than 75% compared with that produced by the initial solid-state fermentation, and the prepared xylanase exhibits excellent acid resistance and high temperature resistance.

[0015] It should be noted that the strain inoculation in the present invention is to prepare the Aspergillus sp. G63 strain to a concentration of 1×10 6The spore suspension with spores / mL was then inoculated into solid fermentation medium.

[0016] The application of the xylanase produced by the method of the invention in catalyzing the hydrolysis of xylan has broad application prospects and promotion value.

[0017] The beneficial effects of the present invention are: The present invention significantly improves the number and vitality of spores by selecting the black Aspergillus strain Aspergillus sp.G63 with high xylanase production, thereby greatly improving production efficiency. Compared with traditional liquid fermentation, the solid fermentation technology adopted by the present invention has obvious advantages such as simple operation and easy acquisition of raw materials. The method cleverly utilizes low-cost solid waste, such as bran and soybean meal, as a carbon source and nitrogen source for bacterial growth, realizes the recycling of resources, and effectively reduces production costs. The present invention selects the relevant influencing factors of the culture medium and culture conditions, and optimizes them through the response surface method to obtain the optimal solid-state fermentation culture medium and culture conditions suitable for the strain of the present invention, and the produced xylanase also has excellent acid resistance and high temperature resistance. In addition, the production process design of the present invention is simple, easy to operate, short fermentation cycle, stable product quality, and high enzyme activity, which makes it very suitable for wide application and promotion in the industrial field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of soybean meal bran ratio on xylanase activity produced by Aspergillus niger G63; Figure 2 The effect of the type of additional nitrogen source on the xylanase activity produced by Aspergillus niger G63; Figure 3 The effect of additional nitrogen source addition on the xylanase activity produced by Aspergillus niger G63; Figure 4 The effect of metal ion types on the xylanase activity produced by Aspergillus niger G63; Figure 5 The effect of metal ion addition on the xylanase activity produced by Aspergillus niger G63; Figure 6 The effect of water content on the activity of xylanase produced by Aspergillus niger G63; Figure 7 The effect of inoculation amount on the xylanase activity produced by Aspergillus niger G63; Figure 8 The effect of loading amount on the xylanase activity produced by Aspergillus niger G63; Fig. 9 The effect of temperature on the xylanase activity produced by Aspergillus niger G63; Fig.10 The effect of pH on the activity of xylanase produced by Aspergillus niger G63; Fig.11 The response surface of the effect of water content and temperature on the activity of xylanase produced by Aspergillus niger G63; Fig.12 The response surface of the effect of water content and inoculum size on the xylanase activity produced by Aspergillus niger G63; Fig.13 The response surface of the effect of temperature and inoculum size on the xylanase activity produced by Aspergillus niger G63; Fig.14 The contour lines of the effects of temperature and water content on the xylanase activity produced by Aspergillus niger G63; Fig.15 The contour lines of the effects of water content and inoculum size on the xylanase activity produced by Aspergillus niger G63; Fig.16 The contour lines of the effects of temperature and inoculation amount on the xylanase activity produced by Aspergillus niger G63; Fig.17 The effect of temperature on the relative enzyme activity of xylanase produced by Aspergillus niger G63 before and after fermentation culture optimization; Fig.18 The pH stability of the effect of pH on the relative enzyme activity of xylanase produced by Aspergillus niger G63 fermentation before and after optimization; Fig.19 TLC analysis of xylan hydrolyzed by xylanase produced for fermentation culture optimization. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to specific embodiments.

[0020] The Aspergillus niger strains used in the following specific examples of the present invention Aspergillus niger G63 was deposited on October 28, 2024 in the General Microbiology Center of China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with the deposit number CGMCC NO.41556.

[0021] Aspergillus niger strain in the present invention Aspergillus niger The activation and solid-state fermentation culture method of G63 is as follows: Aspergillus nigerG63 was deposited with the accession number CGMCC NO.41556. The activated culture was carried out on PDA solid medium (PDA solid medium composition: potato 200 g / L, glucose 20 g / L, agar 20 g / L, water was added to 1000 mL, and sterilized at 115 °C for 30 min) for 5 days. The Aspergillus niger spores were washed off with sterile saline and placed in a sterilized centrifuge tube containing glass beads. The spores were fully shaken to break up the spores. The spore suspension was filtered with sterile gauze. The number of spores was counted using a hemocytometer, and the spores were diluted with saline to a concentration of 1 × 10 6 The spore suspension was inoculated into a sterilized solid fermentation medium, stirred evenly, and then cultured for 5 days to produce xylanase.

[0022] The test method for the enzymatic activity of xylanase in the present invention is the DNS method: Preparation of acetic acid-sodium acetate buffer: add 1.7 mL of glacial acetic acid to 23.14 g of sodium acetate trihydrate, then add water to dissolve, make up to 2000 mL, and adjust the pH to 5.5.

[0023] Preparation of substrate xylan solution: weigh 1.00 g of xylan, add 0.32 g of sodium hydroxide, and then add 90 mL of water, stir magnetically and heat slowly until the xylan is completely dissolved. Stop heating and continue stirring for 30 min, then add 0.5 mL of glacial acetic acid. Continue magnetic stirring, adjust the pH to 5.50 with acetic acid solution or sodium acetate solution, dilute the acetic acid-sodium acetate buffer solution to 100 mL, and shake well before use. Store at 4°C away from light, with a shelf life of 12 hours.

[0024] Preparation of crude enzyme solution: Dry and grind the fermentation wet base after solid-state fermentation, weigh 1.00g of the dried and crushed xylanase fermentation substrate, add 40mL of acetic acid-sodium acetate buffer solution, stir magnetically for 30min, and then dilute to 100mL with buffer solution, and store at 4℃ in the dark for 1h~2h. Centrifuge at 1000g for 3min~5min, take 1.0mL of supernatant, and dilute to 50mL with buffer solution. This is the crude enzyme solution.

[0025] Enzyme activity was determined by DNS method. The reaction system was: 2.00 mL crude enzyme solution, 2.0 mL xylan solution, electromagnetic oscillation for 3s~5s, and precise temperature maintenance at 37℃ for 30 min. Add 5.0 mL DNS reagent and electromagnetic oscillation for 3s~5s to terminate the enzymatic reaction. Heat in a boiling water bath for 5 min, cool to room temperature, add water to make the volume 25 mL, and electromagnetic oscillate for 3s~5s. Measure the absorbance at 540nm. Definition of enzyme activity unit: Under the conditions of 37℃ and pH 5.50, the amount of enzyme required to degrade and release 1μmol of reducing sugar from a xylan solution with a concentration of 5mg / mL per minute is one enzyme activity unit U. Example 1

[0026] A single factor experiment of fermentation medium was conducted, and solid-state fermentation of high-yielding xylanase strains was carried out to investigate the effect of fermentation medium on xylanase.

[0027] (1) Effect of solid matrix ratio on xylanase production by Aspergillus niger Soybean meal and bran were selected as the solid matrix of the culture medium, and different ratios of soybean meal and bran were used, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2. Other initial fermentation conditions were (NH4)2SO4 0.45g, MgCl2 0.33g, H2O 18.6mL, loading amount 30g, natural pH, and static fermentation at 28℃ for 5 days. The xylanase activity was measured, and the results were as follows: Figure 1 As shown. Figure 1 It can be seen that when the ratio of soybean meal to bran is 3:7, the xylanase activity reaches the maximum value. Therefore, the ratio of soybean meal to bran is 3:7 as the solid matrix formula for subsequent experiments.

[0028] (2) Effect of the type of additional nitrogen source on xylanase production by Aspergillus niger On the basis of the optimal solid matrix ratio, urea, (NH4)2SO4, NH4Cl, KNO3, and NaNO3 were used as additional nitrogen sources, and the addition amount was 1.5%. The other conditions were the initial fermentation conditions, MgCl2 0.33g, H2O 18.6mL, charge amount 30g, natural pH, 28℃ static fermentation for 5 days, and the xylanase activity was measured. The results are as follows Figure 2 As shown. Figure 2 It can be seen that when NH4Cl was used as the additional nitrogen source, the xylanase activity reached the maximum value. Therefore, NH4Cl was selected as the best additional nitrogen source.

[0029] (3) Effect of the amount of additional nitrogen source added on xylanase production by Aspergillus niger On the basis of the optimal nitrogen source, in order to further determine the effect of NH4Cl addition on xylanase, five levels of addition were selected for experiment: 0.9%, 1.1%, 1.3%, 1.5%, and 1.7%. Other conditions were initial fermentation conditions: MgCl2 0.33g, H2O 18.6mL, charge amount 30g, natural pH, 28℃ static fermentation for 5 days, and the enzyme activity of xylanase was measured. The results are as follows Figure 3 As shown. Figure 3 It can be seen that the xylanase activity is maximum when the NH4Cl addition amount is 1.3%, so the NH4Cl addition amount is selected to be 1.3%.

[0030] (4) Effect of metal ion types on xylanase production by Aspergillus niger Based on the optimal solid matrix ratio, optimal nitrogen source and its addition amount, K + , Mn 2+ Mg 2+ , Fe 3+ 、Na + The other conditions are the initial fermentation conditions, H2O 18.6mL, charge 30g, natural pH, 28℃ static fermentation for 5 days, and the xylanase activity was measured. The results are as follows Figure 4 As shown. Figure 4 It can be seen that when Na + When used as a metal ion, the xylanase activity reaches its maximum value. Therefore, Na + as metal ions.

[0031] (5) Effect of metal ion addition on xylanase production by Aspergillus niger Based on the optimal solid matrix ratio, optimal nitrogen source and its addition amount, and optimal metal ion, in order to further determine the effect of metal ion addition on the production of xylanase by Aspergillus niger, five levels of addition amount of 0.7%, 0.9%, 1.1%, 1.3%, and 1.5% were selected for experiment. Other conditions were initial fermentation conditions, H2O 18.6mL, charge amount 30g, natural pH, 28℃ static fermentation for 5d, and xylanase activity was measured. The results are as follows Figure 5 As shown. Figure 5 It can be seen that when Na + When the addition amount is 1.1%, the xylanase activity reaches the maximum value. + The addition amount is 1.1%.

[0032] (6) Effect of water content on xylanase production by Aspergillus niger Based on the optimal fermentation medium composition, the xylanase activity was determined at 45%, 50%, 55%, 60%, and 65% water content, 30 g loading, natural pH, and 28°C for 5 days. Figure 6 As shown by Figure 6 It can be seen that when the water content is 60%, the xylanase activity reaches its maximum value. Therefore, the water content is selected to be 60%.

[0033] The best fermentation medium composition obtained above is soybean meal: bran 3:7, the best nitrogen source addition amount is 1.3% NH4Cl, the best metal ion addition amount is 1.1% Na + The optimum moisture content is 60%, and on this basis the fermentation culture conditions are optimized. Example 2

[0034] Single factor test of fermentation culture conditions (1) Effect of inoculation amount on xylanase production by Aspergillus niger Based on the optimal fermentation medium composition and optimal water content, the xylanase activity was determined with an inoculation volume of 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 2.5 mL, a loading volume of 30 g, natural pH, and static fermentation at 28°C for 5 days. Figure 7 As shown by Figure 7 It can be seen that when the inoculation volume is 1.5 mL, the xylanase activity reaches the maximum value. Therefore, the inoculation volume is selected as 1.5 mL.

[0035] (2) Effect of feed amount on xylanase production by Aspergillus niger Based on the optimal fermentation medium composition, optimal water content and optimal inoculum size, the xylanase activity was determined with a loading of 10g, 20g, 30g, 40g and 50g, natural pH, and 28℃ for 5 days. Figure 8 As shown by Figure 8 It can be seen that when the loading amount is 30 g, the xylanase activity reaches the maximum value. Therefore, the loading amount is selected to be 30 g.

[0036] (3) Effect of temperature on xylanase production by Aspergillus niger Based on the optimal fermentation medium composition, optimal water content, optimal inoculation amount and optimal loading amount, the xylanase activity was determined at 22℃, 25℃, 28℃, 31℃ and 34℃, natural pH, and static fermentation for 5 days. Fig. 9 As shown by Fig. 9 It can be seen that when the fermentation temperature is 28°C, the xylanase activity reaches the maximum value. Therefore, the fermentation temperature is selected to be 28°C.

[0037] (4) Effect of pH on xylanase production by Aspergillus niger Based on the optimal fermentation medium composition, optimal water content, optimal inoculation amount, optimal loading amount and optimal culture temperature, the xylanase activity was determined at pH 3.5, 4.5, 5.5, 6.7 and 7.5 for 5 days of static fermentation. Fig.10 As shown by Fig.10 It can be seen that when the pH is 5.5, the xylanase activity reaches its maximum value. Therefore, the pH value of 5.5 was selected. Example 3

[0038] Through the single factor experiment in Example 1, in order to better determine the culture medium components for enzyme production by the strain, three factors, namely nitrogen source addition, water content and metal ion addition, were selected to design an orthogonal experiment of culture medium components. The specific factor levels of the orthogonal experiment are shown in Table 1. The orthogonal table was designed according to the orthogonal design software for the experiment, and the experimental results are shown in Table 2.

[0039] Table 1 .

[0040] Table 2 .

[0041] Through the single factor experiment in Example 2, in order to better determine the culture conditions for enzyme production of the strain, three factors of temperature, inoculation amount and pH were selected to design an orthogonal experiment of culture conditions. The specific factor levels of the orthogonal experiment are shown in Table 3. The orthogonal table was designed according to the orthogonal design software to conduct the experiment, and the experimental results are shown in Table 4.

[0042] Table 3 .

[0043] Table 4

[0044] This example illustrates that the culture medium components that affect the activity of xylanase are, from the most important to the least important, water content, metal ion addition, and nitrogen source addition; and the culture conditions that affect the activity of xylanase are, from the most important to the least important, temperature, inoculation amount, and pH. Example 4

[0045] Through the orthogonal experiment in Example 3, in order to better explore the conditions for enzyme production of the strain, three factors, namely water content, temperature and inoculation amount, were selected to design a response surface optimization experiment. The specific factor levels of the response surface optimization experiment are shown in Table 5. The experimental results were analyzed using the Design-Expert software, as shown in Table 6.

[0046] Table 5 .

[0047] Table 6 .

[0048] As can be seen from Table 6, the fitting equation obtained by the response surface experiment is: Y = 26638.3 + 238.12A +1049.62B + 477.75C - 350AB - 885.25AC + 272.25BC - 2692.92A² - 4437.42B² -2756.67C². The F value in the model equation is 365.78, and the P value is <0.0001, which meets the significant condition of the model. The regression equation determination coefficient R 2 =0.9985, and Adj=0.9958, indicating that the model fits the experiment well, the experimental error is small, and it can better reflect the accurate relationship between the three factors and xylanase in the fermentation process. The influence of each factor on xylanase activity is B>C>A. Figure 11-16As shown in the figure, AB, AC, and BC all have obvious interactions, especially AC. Therefore, the optimal fermentation medium for enzyme production of the mutant strain Aspergillus niger is: 21g bran, 9g soybean meal, 1.5% NH4Cl addition, and 1.5% Na + The addition amount was 1.1%, the water content was 60%, the inoculation amount was 1.5 mL, the loading amount was 30 g, the pH was 5.5, the fermentation temperature was 28 ° C, and the culture time was 5 days. Example 5

[0049] In order to further investigate the effect of the optimized fermentation medium on the properties of xylanase, the Aspergillus niger strain Aspergillus niger G63 (CGMCC NO.41556) produced xylanase under the optimal fermentation conditions: bran 21g, soybean meal 9g, NH4Cl addition 1.5%, Na + The addition amount was 1.1%, the water content was 60%, the inoculation amount was 1.5mL, the loading amount was 30g, the pH was 5.5, the fermentation temperature was 28℃, and the culture time was 5d. The initial solid fermentation medium was used as a control, and 1.0mL of the spore suspension of Aspergillus niger was inoculated in the initial solid fermentation medium (23.4g bran, 6.6g soybean meal, 0.45g (NH4)2SO4, 0.33g MgCl2, 18.6mL H2O, sterilized at 121℃ for 20min), and fermented at 28℃ for 5 days. The xylanase measured after fermentation of the strain under the optimized optimal conditions was 26901 U / g dry basis, which was 75.1% higher than that before optimization (15365U / g dry basis), which greatly explored the production potential of the strain, achieved the ideal goal, and contributed to the industrial application of xylanase.

[0050] The crude enzyme solution was precipitated with ammonium sulfate, concentrated by dialysis, filtered by membrane, and then loaded on an anion exchange column (DEAE Beads6FF) for chromatography. Gradient elution was performed to obtain purified xylanase. The enzyme activity was detected by DNS method and diluted with acetic acid-sodium acetate buffer solution to a concentration of 100 U / mL.

[0051] The enzymatic properties of xylanase produced by Aspergillus niger G63 after response surface methodology optimization were compared with those produced by initial solid-state fermentation medium fermentation. The results are as follows: like Fig.17 As shown in the figure, the relative enzyme activity of the optimized Aspergillus niger G63 xylanase reaches a peak at 65°C, which has a certain heat-resistant advantage and is a thermostable xylanase. As the temperature further increases, the relative enzyme activity shows a downward trend. This is mainly because high temperature will destroy the protein structure of xylanase, resulting in a decrease in its catalytic activity. Nevertheless, within the temperature range of 35°C to 85°C, the relative enzyme activity of xylanase can still be maintained above 85%.

[0052] like Fig.18 As shown in the figure, the relative enzyme activity of the xylanase produced by the optimized Aspergillus niger G63 reached a peak at pH 4.0. In the pH range of 2.5 to 7.5, the relative activity of the enzyme can be maintained at more than 85%. This shows that the xylanase produced by the optimized Aspergillus niger G63 has a good adaptability to the acidic environment and is an acid-resistant xylanase. Changes in pH value will affect the charge state of the polar side chains in the enzyme molecule, causing changes in the ionization state of these groups, thereby changing the structure of the enzyme active site. This structural change will affect the spatial conformation of the protein, ultimately leading to protein denaturation and inactivation, thereby reducing the catalytic efficiency of the enzyme. Therefore, after reaching the optimal pH, the relative enzyme activity of the xylanase will gradually decrease as the pH further increases.

[0053] 1 mL of the purified 100 U / mL xylanase solution and 1 mL of the xylan solution were reacted at 50°C for 8 hours, and the product components were analyzed by thin layer chromatography (TLC). Fig.19 As shown, the purified xylanase can effectively hydrolyze xylan into xylose within 8 h, and the hydrolysis product of the xylanase produced by Aspergillus niger G63 after optimization is darker in color on the silica gel plate than before optimization, indicating that its hydrolysis ability is stronger.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. For a person skilled in the art, any obvious changes made thereto without departing from the principle and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for producing xylanase by fermentation of Aspergillus niger optimized based on response surface methodology, characterized in that: Using the Aspergillus sp. G63 deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) on October 28, 2024, with a deposit number of CGMCC NO.41556 as the fermentation strain, inoculating the solid fermentation medium to produce xylanase, comprising the following specific steps: S1 Single factor experiment to determine the solid fermentation medium and fermentation culture conditions; S2 orthogonal experiment to determine the most relevant influencing factors of solid fermentation medium and fermentation culture conditions; S3, based on orthogonal experiments, selected the most relevant influencing factors and used response surface methodology to optimize solid-state fermentation medium and fermentation culture conditions; S4 produced xylanase under the solid-state fermentation medium and fermentation culture conditions optimized by response surface methodology.

2. A method for producing xylanase by fermentation of Aspergillus niger optimized based on response surface methodology, characterized in that: The solid-state fermentation medium described in step S1 includes a carbon source, a nitrogen source, an additional nitrogen source, metal ions and water; the carbon source is bran, and the nitrogen source is soybean meal; the experimental factors of the single-factor experiment include the ratio of bran to soybean meal in the solid-state fermentation medium, the type and addition amount of the additional nitrogen source, the type and addition amount of metal ions, and water content; as well as the temperature, pH, charge amount, and inoculation amount of the fermentation culture.

3. A method for producing xylanase by fermentation of Aspergillus niger optimized based on response surface methodology, characterized in that: In step S2, a three-factor three-level design is used to determine the most relevant influencing factors of the solid-state fermentation medium composition and the fermentation culture conditions respectively; wherein the experimental factors of the solid-state fermentation medium composition are the additional nitrogen source addition amount, water content and metal ion addition amount; and the experimental factors of the fermentation culture conditions are temperature, inoculation amount and pH.

4. A method for producing xylanase by fermentation of Aspergillus niger optimized based on response surface methodology, characterized in that: The experimental factors in step S3 are moisture content, temperature and inoculum size.

5. A method for producing xylanase by fermentation of Aspergillus niger optimized based on response surface methodology, characterized in that: The solid-state fermentation medium and fermentation culture conditions optimized by response surface methodology were as follows: 21 g bran, 9 g soybean meal, 1.5% NH4Cl addition, and 1.5% Na + The addition amount is 1.1%, the water content is 60%, the inoculation amount is 1.5 mL, the loading amount is 30 g, the pH is 5.5, the fermentation culture temperature is 28 ° C, and the culture time is 5 days.

6. Use of the xylanase produced by the method according to any one of claims 1 to 5 in catalyzing xylan hydrolysis reaction.

7. The use according to claim 6, characterized in that: When the xylanase catalyzes the hydrolysis of xylan, the pH of the reaction system is 2.5-7.5 and the reaction temperature is 35-85°C.

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